Difference in hepatic uptake of tetra- and Di-bromosulfophthalein in rat: Role of hydrophobicity, binding to plasma proteins and affinity for plasma membrane carrier protein

Abstract

The relative role of hydrophobicity, binding to plasma proteins and affinity for one of the plasma membrane transport proteins in the hepatic uptake of 3,4,5,6-tetra- (BSP) and 3,6-di- (DBSP) bromosulfophthalein was investigated in the rat. In terms of physicochemical characteristics, the two molecules show different p K a , values and degrees of hydrophobicity, as determined from the n-octanol: water partition coefficient. In the intact animal, the plasma clearance and the plasma removal rate after a dose of 1.5 μmol/kg i.v. were significantly (P < 0.001) faster for BSP than DBSP, while no difference was found in the plasma distribution volume. The dissociation constant ( K d ) of the high affinity binding sites of plasma proteins also differed for the two anions, being significantly lower for BSP than DBSP (0.95 ± 0.02 vs 1.44 ± 0.14 μM, P < 0.001). [ 35S]BSP uptake by liver plasma membrane vesicles was saturable with an apparent K m of 5.20 ± 0.80 μM, and was competitively inhibited by DBSP ( K i 18.2 ± 1.2 μM) indicating a common uptake system. The K d value for binding of the organic anions to purified bilitranslocase, a plasma membrane protein involved in the electrogenic transport of pthaleins, was also significantly lower for BSP than DBSP (1.10 ± 0.12 vs 3.02 ± 0.27 μM, N = 3, P < 0.001), indicating a higher affinity of the former ligand for the carrier protein. No difference was observed in the capacity of the high affinity binding sites (32 ± 3 vs 33 ± 3 nmol/mg protein, BSP and DBSP, respectively). These data indicate that BSP and DBSP are two different cholephilic organic anions which share a common uptake mechanism, at least partly mediated by bilitranslocase. The greater affinity of BSP than DBSP for the carrier protein may account for the faster plasma disappearance rate of BSP observed in vivo, in spite of the higher plasma protein binding.